Major histocompatibility antigens (MHC) are highly polymorphic, and thus possess numerous antigenic determinants. These antigens are essential for function of cytotoxic T lymphocytes. Our basic approach to the understanding of structure-function relationship has been to alter the structure of the genes that encode mouse Class I (H-2) MHC antigens in vitro and then to examine the structural basis for functions involved in T cell recognition and immunological polymorphism. The polymorphism can be dissected by a series of monoclonal antibodies which we produced previously. In the past year we have isolated several mouse Class I genes from the BALB/c (H-2d haplotype) genome. A Class I protein is mainly composed of 3 discrete domains which correspond to 3 main exons interspersed by introns in the gene. More recently, we have produced hybrid H-2 genes in which the C2 domain (closer to the membrane) has been exchanged between the two polymorphic genes (H-2Ld and H-2Dd). Studies of the products of hybrid genes have revealed that polymorphic sites recognized by monoclonal antibodies and by a variety of cytotoxic T cells are predominantly localized in the first two external domains, i.e. N and/or C1. Currently the exchange of N or C1 domains between two genes of different haplotypes (H-2d and H-2p) is underway using techniques of molecular genetics. Further, in vitro mutagenesis of the H-2 gene is being undertaken by alteration of a single amino acids at various desired positions. We apply the method of oligonucleotide-directed site-specific mutagenesis for this study. Alteration of glycosylation sites, disruption of a disulfide bond, and single amino acid substitutions at other positions expected to be important for conferring antigenicity are planned in this mutagenesis scheme. These new genes will be introduced into mouse L cells by DNA-mediated gene transfer, and effect upon discreet immunological functions will be studied.